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International Immunology Advance Access originally published online on March 15, 2007
International Immunology 2007 19(4):447-453; doi:10.1093/intimm/dxm009
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© The Japanese Society for Immunology. 2007. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Both Gimap5 and the diabetogenic BBDP allele of Gimap5 induce apoptosis in T cells

Ulla Dalberg, Helle Markholst and Lars Hornum

Department of Immunopharmacology, Hagedorn Research Institute, Novo Nordisk Park F6.2.30, DK-2760 Måløv, Denmark

Correspondence to: H. Markholst; E-mail: hmar{at}hagedorn.dk


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Gimap5, a member of the GTPase of the immunity-associated protein family (Gimap), regulates T cell survival. A strong indication of this is found in the diabetes-prone BioBreeding rat (BBDP), where a frameshift mutation in Gimap5 results in T-cell lymphopenia. We have investigated the function of human Gimap5 in T cells. We found that reduction of Gimap5 by RNA interference in Jurkat cells did not affect the number of apoptotic cells whereas transient over-expression of Gimap5 resulted in a major increase in the number of apoptotic cells. The same effect of over-expression was found in naive human T cells purified from blood but not in activated human T cells. This suggests that the apoptosis-inducing effect of Gimap5 over-expression is dependent on the activation status of the cells. Since the apoptosis-inducing effect of Gimap5 was contrary to the expected function of Gimap5 based on the phenotype of BBDP rats, we over-expressed rat wt Gimap5 and Gimap5 with the mutation found in BBDP (Gimap5-lyp). Both versions of rat Gimap5 induced apoptosis when expressed in the rat T-cell line C58(NT)D.1.G.OVAR.1, however, Gimap5-lyp greatly exacerbated cell death. Finally, we detected the subcellular localization of Gimap5 to be at the endoplasmic reticulum and by quantitative PCR, we found that endogenous Gimap5 mRNA is up-regulated in activated T cells.

Keywords: diabetes-prone BB rat, Jurkat, Lyp, T cells


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 Supplementary data
 References
 
Gimap5 (Gimap, GTPase of the immunity-associated protein family) is a member of a family of seven Gimap genes in humans and rats and eight genes in mice. Members of the family are found in all vertebrates examined so far and in several plants (1). The AIG-1 domain—a GTP-binding domain—is contained in all Gimap genes and the Gimap genes are mainly expressed in thymocytes and lymphocytes (1–3). Although the genes are largely uncharacterized, several reports suggest an important function in regulation of apoptosis in lymphocytes (1, 4–9). Further support for this is that the genes are down-regulated in diverse cancer tissues and cell lines of both human and rat origin (2, 3) and that the Gimap5 family is located within a region on chromosome 7q35–q36 which is frequently deleted in acute myeloid leukemias (10). Recent published papers suggest a function of the Gimap genes in thymocyte maturation and selection, since the genes are differentially expressed in both mice and rat thymocyte subsets (1, 2). Reduction of Gimap5 in fetal thymus organ cultures by RNA interference (RNAi) changes the ratio of double negative and double positive thymocytes (1) which strongly suggest a function of Gimap5 in thymocyte selection in mice.

Naive T-cell homeostasis is maintained by a delicate balance between thymic output, proliferation, differentiation and apoptosis. Long-term survival of T cells is tightly controlled by TCR signaling due to recognition of self-ligands bound to MHC molecules, and IL-7 is necessary as a co-stimulatory signal (11). In the diabetes-prone BioBreeding rat (BBDP), homeostasis of the naive T cells is dysregulated resulting in a severe T-cell lymphopenia that segregates as a single, fully penetrant and recessive trait in crosses between BBDP and the non-lymphopenic diabetes resistant BioBreeding rat (BBDR) (12, 13). The recent thymic emigrant cells in BBDP die by apoptosis within 1 week after entering the circulation unless rescued by activation through the TCR (14). A positional cloning effort to detect the genetic defect (lyp) causing the life-long T-cell lymphopenia in BBDP led to the identification of a frameshift mutation in Gimap5 (15, 16). The frameshift mutation results in a truncated protein where the last 224 amino acids (AA) of the 308 AA long wild-type (wt) Gimap5 protein are replaced by 19 other AA. A 150-kb P1 artificial chromosome transgene harboring a wt allele of the rat Gimap5 gene as well as other Gimap genes restored Gimap5 transcript and protein levels as well as normal T-cell numbers in a F344 rat congenic for the T-cell lymphopenia locus from BBDP, F344lyp/lyp (17). The phenotype of lyp/lyp rats suggests that Gimap5 also plays a role in regulation of T-cell activation because the recent thymic emigrants are semi-activated. They have activated nuclear factor-kappa B and they can be activated by anti-CD3 stimulation without the need for co-stimulation (18–20).

We set out to characterize Gimap5. We report here the effect of reduction of human Gimap5 by RNAi in Jurkat cells and the effect of over-expression of Gimap5 in Jurkat and primary T cells. We report the effect of transient over-expression of rat Gimap5-wt and rat Gimap5 with the BBDP mutation (Gimap5-Lyp). Moreover, we have investigated the endogenous expression of Gimap5 in naive and activated human T cells and the subcellular localization of the protein.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 Supplementary data
 References
 
Cell lines and human primary T cells
Jurkat cells clone E6-1 and C58(NT)D.1.G.OVAR.1 (C58) cells (American Type Culture Collection, LGC Promochem, Boras, Sweden) were grown at 37°C and 5% CO2 in supplemented medium according to the manufacturer's manual. Cell numbers were kept between 105 and 106 cells ml–1 and no stock was kept in culture for >10 passages.

Human primary CD3+ cells were purified from blood (<2 h old). Firstly, PBMC were purified by centrifugation in Vacutainer CPT tubes (BD Biosciences, Erembodegem, Belgium). Then, CD3+ cells were purified from the PBMC using Pan T cell isolation kit II (Miltenyi, Bergisch Gladbach, Germany) resulting in a non-magnetic, untouched cell fraction of ≥95% pure CD3+ cells. Primary T-cell cultures were started with a concentration of 106 cells ml–1. The naive T cells were cultured in RPMI with Glutamax (Invitrogen, Taastrup, Denmark) with 10% human serum type AB (Sigma, MO, USA). For activation of T cells, the freshly purified T cells were cultured for 17 h in complete-RPMI-10 (human serum type AB) supplemented with 10 µg ml–1 PHA and 4 ng ml–1 IL-2 which equals ~100 U ml–1 (Cat. no. 554603, BD Biosciences). The cells were then washed twice followed by culture in complete-RPMI-10 with or without 4 ng ml–1 IL-2. If the cells were cultured for >4 days, the medium was changed at day 4.

Plasmids and transfections
The RNAi vectors were made by insertion of the Gimap5-specific hairpin sequence 5'-GCGCTCCTCAGAGTCAAATTCAAGAGATTTGACTCTGAGGAGCGCCTTTTT-3' or the negative control sequence 5'-CTCGGCCGAGATACTAACTTCAAGAGAGTTAGTATCTCGGCCGAGCTTT-TT-3' into psiSTRIKE-hMGFP (Promega, Ramcon, Birkerød, Denmark). The negative control sequence does not contain high homology to any genes at the National Center for Biotechnology Information Genbank, tested by the BLAST function. The CMW promoter and MGFP gene in psiSTRIKE-hMGFP were replaced by the CMW promoter and EGFP gene from pEGFP-C1 (BD Biosciences) in order to obtain satisfactory green fluorescence in the transfected cells. Three independent plasmid preparations of each construct were made with Endofree MaxiPrep (Qiagen, West Sussex, UK).

Human and rat Gimap5 were cloned into pEGFP-C1 (BD Biosciences). Full-length human Gimap5 was amplified by PCR from Jurkat cDNA with the primers: 5'-ATATGAATTCACAACAACCGTTTCT-GGAGA-3' and 5'-ACGAGGATCCATTAGAAGTGCTCCAGGGTC-3'. Highlighted in bold are the restriction enzyme sites for EcoRI and BamHI, which were hereby added to the PCR product. Full-length rat Gimap5-wt and full-length rat Gimap5-lyp were amplified by PCR from BBDR cDNA and BBDP cDNA, respectively, using the primers: 5'-ATGATGAATTCGAAGCTGCGGTGCACTC-3' and 5'-ATGTGCGCCGCGGTTGGTGGACAGGGATAATGGC-3'. Highlighted in bold are the restriction enzyme sites for EcoRI and SacII, which were hereby added to the PCR products. The resulting plasmids which encode a protein with EGFP linked N-terminally to Gimap5 were analyzed for correct cloning by sequencing (performed by MWG Biotech, Martinsried, Germany). Three independent plasmid preparations of each construct were made with Endofree MaxiPrep (Qiagen).

Jurkat cells and C58 cells were transfected with Nucleofector (Amaxa, Reactionlab, Lynge, Denmark) using Cell line nucleofector kit V and transfection program G-10. Freshly purified T cells were transfected similarly using the human T cell nucleofector kit and transfection program U-14. All transfections were performed with 3 x 106 cells and done according to the manufacturer's manual. Jurkat transfections for RNAi studies were done with 2.5 µg vector. Jurkat and C58 transfections for over-expression studies were done with 4 µg pEGFP or 4.8 µg pEGFP-Gimap5 in order to transfect with the same number of plasmids (1.3 x 10–12 moles). All primary T-cell transfections were done with 2 µg pEGFP or 2.4 µg pEGFP-Gimap5. All transfections were performed in triplicate with three independent plasmid preparations. Two-tailed Student's t-test was used for all statistical analysis of results.

Flow cytometry
Flow cytometry was performed on a FACSCalibur (BD Biosciences). All antibodies used were from BD Biosciences. The purity of the CD3+ cells was assessed with anti-CD3 (clone HIT3a) antibodies and the activation was assessed with anti-CD69 (clone FN50) and anti-CD25 (clone M-A251) antibodies. Apoptotic cells were detected by staining with AnnexinV–allophycocyanin and 7-amino-actinomycin-D (7AAD). AnnexinV binds to phosphatidylserine on apoptotic and dead cells and 7AAD binds to the DNA of dead cells. Apoptosis was also detected by staining with the mitochondrial dye tetramethylrhodamine methyl ester (TMRM) (Invitrogen) which only stains intact mitochondria.

Quantitative PCR
Human primary CD3+ cells were purified from blood as described above. The cDNA was made with SuperScript II (Invitrogen) from total RNA made with Absolutely RNA Microprep kit (Stratagene, Amsterdam, The Netherlands). The PCRs were performed with [32P]-dCTP and aliquots were taken out at N, N + 2 and N + 4 cycles and loaded on standard sequencing gels (Gel-Mix 6%; USB, Cleveland, OH, USA). Primers for the reaction were 5'-TGAGTCACAGGCCGATACCC-3' and 5'-TTCTCTCACACTCCCGCACCA-3'. QuantumRNA Classic II 18S (Ambion, Cambridgeshire, UK), which amplifies 18S ribosomal RNA, were used for normalization with the ratio of 1:9 of 18S-primers:competimers. The specific bands were then quantified using a Typhoon 8600 variable mode imager (GE Healthcare, Hillerød, Denmark). Quantifications were made when both Gimap5 and the control gene were in the exponential phase. Three different samples of cells were analyzed.

Detection of subcellular localization by confocal microscopy
For staining of the mitochondria, the cells were incubated in cell culture medium containing 50 nM MitoFluor Red 589 (Invitrogen) for 30 min at 37°C followed by centrifugation and re-suspension in cell culture medium. For staining of the endoplasmic reticulum (ER), the cells were incubated in cell culture medium containing 500 nM ER-Tracker Blue–White DPX (Invitrogen) and incubated for 30 min at 37°C followed by centrifugation and re-suspension in cell culture medium. The cells were immediately analyzed on a Zeiss confocal microscope model LSM 510 META.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 Supplementary data
 References
 
Reduction of Gimap5 via RNAi had no effect on apoptosis in Jurkat cells
The effect of reduced amounts of Gimap5 was tested by RNAi. Jurkat cells were transfected with a Gimap5-specific RNAi vector (Gimap5-RNAi) or a control vector (control-RNAi). The FACS analyses of the cells were exclusively made on transfected cells (EGFP+). Transfection with Gimap5-RNAi resulted in 77 ± 4% reduction of the Gimap5 mRNA compared with control-RNAi or mock transfected cells (quantified by PCR). Reduction of Gimap5 did not affect the number of apoptotic (Fig. 1A) or dead cells. The level of EGFP fluorescence reflects the number of copies of the vector that the individual cell has received. It is therefore plausible to assume that the cells with a high EGFP expression have a higher reduction of Gimap5 mRNA than the average of 77%. However, also when the FACS analysis was made exclusively on cells with a high EGFP expression, we did not detect any changes in the number of apoptotic cells (data not shown).


Figure 1
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  		Fig. 1. Apoptotic cells in Gimap5-RNAi transfected Jurkat cells and Gimap5-over-expressing Jurkat cells. Apoptotic cells were detected as AnnexinV+ cells of EGFP+7AAD– cells. The experiment was performed in triplicate and bars indicate standard deviations. (A) Cells transfected with control-RNAi or Gimap5-RNAi vectors. (B) Cells transfected with pEGFP or pEGFP-Gimap5 (*P < 0.001). The absolute numbers of dead and apoptotic cells can be found in Supplementary table 1, available at International Immunology Online.

 
Over-expression of Gimap5 induces apoptosis in Jurkat cells
Jurkat cells were transfected with a plasmid encoding a fusion protein of EGFP and Gimap5 (pEGFP-Gimap5) or a control vector encoding EGFP (pEGFP). On average, a 7-fold increase of Gimap5 expression was detected in the transfected cells. A large increase in the number of apoptotic EGFP+ cells was detected 1 day (2-fold increase) and 2 days (4-fold increase) after the transfection (Fig. 1B). Apoptosis was also measured by TMRM which stains mitochondria of healthy cells. This method revealed the same increase in apoptosis in Gimap5-expressing cells (P = 0.002, data not shown). The increase in apoptosis was accompanied by a reduction in the percentage of live EGFP+ cells. Five days after the transfection, only 1% of the live cells were EGFP+ in cells transfected with pEGFP-Gimap5 whereas 11% of the cells transfected with pEGFP were still EGFP+.

Over-expression of Gimap5 induces apoptosis in naive but not in activated T cells
We examined the effect of over-expression of Gimap5 in naive and activated human T cells to test whether Gimap5 also induced apoptosis in primary cells and whether the activation status of the cells affected the outcome. On average, a 20-fold increase of Gimap5 expression was detected in both the naive and the activated transfected cells. Over-expression of Gimap5 in naive T cells cultured in RPMI-10 induced 2-fold more apoptosis (Fig. 2A) compared with cells transfected with the control vector (pEGFP). For analysis of Gimap5 in activated T cells, transfected cells were activated by PHA and IL-2. The analyses were made exclusively on CD25+EGFP+ cells. Over-expression of Gimap5 in activated cells did not exacerbate apoptosis (Fig. 2B). At the end of a T-cell response, the majority of T cells die by apoptosis likely due to lack of cytokines (21). We tested whether cytokine deprivation of activated cells influenced the effect of Gimap5 over-expression by culturing the cells without IL-2 after the 17 h of activation. As shown in Fig. 2(C), over-expression of Gimap5 did not exacerbate apoptosis.


Figure 2
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  		Fig. 2. Apoptotic cells in Gimap5-over-expressing human T cells. T cells were purified from blood on day 0. All experiments were performed in triplicate and bars indicate standard deviation. (A) Effect of over-expression in naive cells. Apoptotic cells were detected as AnnexinV+ cells of CD25–EGFP+7AAD– cells. (B) Effect of over-expression in PHA and IL-2-activated cells. Apoptotic cells were detected as AnnexinV+ cells of CD25+EGFP+7AAD– cells. (C) Effect of over-expression in PHA–IL-2-activated cells cultured without IL-2 after the activation procedure. Apoptotic cells were detected as AnnexinV+ cells of CD25+EGFP+7AAD– cells (*P = 0.01, **P < 0.01). The absolute numbers of dead and apoptotic cells can be found in Supplementary table 1, available at International Immunology Online.

 
Over-expression of rat Gimap5 with the lyp mutation causes a rapid death of the cells
The frameshift mutation in Gimap5 in BBDP results in a truncated protein which lacks the transmembrane region, a region with coiled-coil structure and part of the AIG domain. In addition 19 AA with no homology to wt-Gimap5 is added due to the frameshift (Fig. 3A). C58, a rat T-cell line, was transfected with a plasmid encoding a fusion protein of EGFP and rat wt Gimap5 (pEGFP-Gimap5-wt), rat Gimap5 with the Lyp mutation from BBDP (pEGFP-Gimap5-Lyp) or a control vector encoding EGFP (pEGFP). Transfection with pEGFP-Gimap5-wt resulted in an exacerbation of apoptosis 48 h after transfection (Fig. 3B). This suggests that the function of Gimap5 is conserved between rats and humans. Transfection with pEGFP-Gimap5-Lyp resulted in very few EGFP+ cells 1 day after transfection most of which were dead. This suggested that expression of Gimap5-Lyp causes a very rapid death. Therefore, we tested the number of apoptotic cells 5 h after transfection (Fig. 3B) and found a significant increase in apoptosis in Gimap5-Lyp-expressing cells. The number of dead EGFP+ cells was significantly increased in cells transfected with pEGFP-Gimap5-wt and pEGFP-Gimap5-Lyp both 5 and 24 h after transfection (Fig. 3C) which further supports the death-inducing function of Gimap5.


Figure 3
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  		Fig. 3. Effect of wt Gimap5 and Gimap5-lyp over-expression in rat C58 cells. (A) Schematic drawing of rat wt Gimap5 and Gimap5-lyp (TM, transmembrane region). (B) Cells transfected with pEGFP, pEGFP-Gimap5-wt or pEGFP-Gimap5-lyp. Apoptotic cells were detected as AnnexinV+ cells of EGFP+7AAD– cells. The experiment was performed in triplicate and bars indicate standard deviations (*P < 0.005, **P < 0.05). (C) Representative dot-plots showing dead EGFP+ cells at 5 and 24 h after transfection with pEGFP, pEGFP-Gimap5-wt or pEGFP-Gimap5-lyp. The percentages in the quadrants are of all cells. The absolute numbers of dead and apoptotic cells can be found in Supplementary table 1, available at International Immunology Online.

 
Gimap5 is up-regulated during T-cell activation
The endogenous Gimap5 expression was examined in Jurkat, naive human T cells and activated human T cells by quantitative PCR normalized to 18S rRNA. The level of Gimap5 mRNA in the naive T cells did not change significantly during the 4 days of culture (Fig. 4). An ~8-fold increase in Gimap5 mRNA levels was detected in activated T cells at days 4 and 7 when compared with the same cells before activation at day 0 (Fig. 4). Jurkat cells were found to express ~2-fold higher levels of Gimap5 than freshly isolated naive T cells but markedly less than activated T cells (Fig. 4).


Figure 4
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  		Fig. 4. Expression level of endogenous Gimap5 in Jurkat and naive or activated human T cells. Gimap5 mRNA was detected by quantitative PCR normalized to 18S RNA. The T-cell cultures and PCRs were made in triplicate and bars indicate standard deviations. Primary human T cells were purified from blood on day 0. The T cells were cultured under two different conditions: (i) without any cytokines (the naive T cells) and (ii) with PHA and IL-2 for 1 day followed by culture with IL-2 (the activated T cells). The level of Gimap5 in the primary cells is set to one at day 0 and the expression on the following days are shown relative to the same cells at day 0. ND, not determined.

 
Gimap5 is located at the ER
We used the plasmid encoding the fusion protein of EGFP and human Gimap5 to study the subcellular localization in human primary T cells using a confocal microscope. We detected no co-localization of EGFP-Gimap5 with the mitochondrial marker, MitoFluor Red 589 (Fig. 5). Instead, we found a clear co-localization with the ER-specific dye, ER-Tracker Blue–White DPX. The ER-Tracker also stains the nuclear membrane as these two organelles have continuous membranes and EGFP-Gimap5 was also detected at the nuclear membrane. We did not detect EGFP-Gimap5 in organelles which were not stained by the ER-Tracker. We conclude that Gimap5 is localized at the ER and the nuclear membrane in human primary T cells although small amounts in the mitochondria cannot be ruled out. This localization was found in both naive and activated T cells and in Jurkat cells (data not shown). When the cells were transfected with the control vector (pEGFP), EGFP was found in the cytoplasm (Fig. 5).


Figure 5
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  		Fig. 5. Subcellular localization of Gimap5. Purified human T cells were transfected with pEGFP-Gimap5 or pEGFP and stained with MitoFluor Red 589 or ER-Tracker Blue–White DPX (shown as red at the pictures) and analyzed by confocal microscopy.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
Supplementary data
 References
 
We have shown here that Gimap5 reduction by RNAi did not affect the number of apoptotic cells. On the contrary, Gimap5 over-expression increases the apoptotic population in Jurkat cells and naive primary T cells but not in activated T cells. We transfected a rat T-cell line with rat wt Gimap5 and Gimap5-Lyp. Both exacerbated apoptosis where the effect of the latter was very rapid and strong. We investigated the expression level of endogenous Gimap5 in naive and activated T cells and found an increase in activated cells when they had been cultured for 4 days. Finally, the subcellular localization of Gimap5 was found to be at the ER.

The BBDP rat has a high increase in apoptosis among the T cells and the interpretation of the BBDP phenotype has been that wt Gimap5 is anti-apoptotic. It has not previously been tested whether the protein product of Gimap5-Lyp has a function. Our results suggest that the T lymphocytes in BBDP die due to the presence of Gimap5-Lyp. This implies that wt Gimap5 is not anti-apoptotic, and indeed we have detected a death-inducing function of wt Gimap5. The lymphopenia in the BBDP rat is inherited as a recessive trait (13) but some changes are seen in the thymic population of F344+/lyp rats (22). The recessive inheritance of lymphopenia and our detection of a death-inducing function of Gimap5-lyp may appear conflicting. An explanation could be that the level of Gimap5-lyp might be low in +/lyp rats perhaps due to a lower half-life of the protein. The Gimap5-lyp protein is severely truncated and it is therefore likely that both the half-life, folding and binding capacities can be altered. Binding of wt Gimap5 to molecular partners in +/lyp cells might override binding of the small amounts of Gimap5-lyp and thereby reduce the effect of Gimap5-lyp. This could explain the lack of lymphopenia in +/lyp rats.

The effect of Gimap5 over-expression was different in naive and activated T cells. Over-expression in naive T cells resulted in an increase in apoptosis. We did not detect an up-regulation of endogenous Gimap5 in naive T cells cultured without supplements. This indicates that Gimap5 is not the cause of neglect-induced death of naive T cells despite its death-inducing capacity in these cells. The activated T cells did not undergo apoptosis when Gimap5 was over-expressed. We detected a high expression of endogenous Gimap5 in activated human T cells which suggests that Gimap5 plays a role in these cells without causing death. A difference in the survival of naive and activated T cells is also detected in the BBDP rat. The naive T cells cannot survive for more than a few days whereas activated and memory T cells have a normal life span.

We found that Gimap5 is localized at the ER in Jurkat cells and naive and activated human T cells. The subcellular localization of human Gimap5 has previously been reported to be at the mitochondria (23) and at the ER, Golgi and centrosomes (8). All other human Gimap proteins where the localization has been tested so far (Gimap1, Gimap4 and Gimap7) are reported to be located at the ER–Golgi (3, 4). The major functions of the ER are transport/folding of proteins and calcium regulation in response to stimulating signals and apoptosis-inducing signals. Gimap5 regulates apoptosis and data from lyp/lyp rats suggest an additional function of Gimap5-lyp in T-cell activation (18–20). This together with the fact that it is localized at the ER could suggest a function of Gimap5 in calcium regulation. Many of the proteins involved in regulation of apoptosis can be found associated with the ER, including, Caspase-12 and Bcl-2 (24). Gimap5 can bind to Bcl-2 and Bcl-xL (1), however, the consequences of these interactions are unknown.

The present study shows that Gimap5 reduction by RNAi in Jurkat cells does not affect the number of apoptotic cells. Unfortunately, we were not able to shut down the expression completely but it has been shown that only 50% reduction can be sufficient for a high reduction at the protein level (1) and we obtained ~80% reduction. A previously reported study by Pandarpurkar et al. (25) showed an increase in apoptosis by Gimap5-RNAi in Jurkat cells. We have tested the same small interfering RNAs as reported by Pandarpurkar et al. (25). We obtained ~50% reduction in mRNA with these probes but we did not detect induction of apoptosis (data not shown). Two other studies with Gimap5-RNAi in lymphoid cell lines also failed to detect any increase in apoptosis in itself (1, 8). Apoptosis was only increased in RNAi-treated cells after addition of unphysiological apoptosis-inducing factors (8) or removal of essential cytokines (1). The latter experiment by Nitta et al. (1) was done in mice and mice differ from rats and humans by the presence of Gimap3, which is highly homologous to Gimap5.

Jurkat cells stably expressing Gimap5 have previously been made by Sandal et al. (8) without any reported increase in apoptotic cells. The lack of apoptosis induction in these cells may be explained by different levels of over-expression in stably transfected cells and our transiently transfected cells. Since high over-expression of Gimap5 induces apoptosis, only the cells with a low over-expression would survive during the preparation of a stable cell line.

The studies by Nitta et al. (1) and Sandal et al. (8) have supported the notion that Gimap5 is anti-apoptotic, our results show a pro-apoptotic function of Gimap5. These results may appear to be conflicting. There are, however, several well-described examples of genes that in some instances induce apoptosis and in others protect against apoptosis. Bcl-2 was discovered as an anti-apoptotic protein, however, transient over-expression of Bcl-2 can induce apoptosis (26). Other examples of this are the proto-oncogenes c-Myc (27) and Ras (28) and cdk inhibitors such as p21 and p27 (29). High expression of proto-oncogenes induces, in most cases, proliferation; however, if the conditions are sub-optimal for proliferation—for example lack of cytokines—the cells undergo apoptosis instead. Thus, the classification of a gene as being either pro- or anti-apoptotic can be a simplification. The effect of Gimap5 appears to be dependent at the activation status of the cells, the availability of growth factors and the presence of toxic compounds. Moreover, the results suggest that the level of Gimap5 needs tight regulation since both a lowering and an increase can be toxic to the cells.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary Table 1 with absolute numbers of the dead and apoptotic cells in figure 1, 2 and 3 is available at International Immunology Online.


   Acknowledgements
 
A Medical Valley Academy Ph.D. grant (U.D.) and an EFSD/JDRF/Novo Nordisk grant (U.D. and L.H.) supported this work. We thank laboratory technician Rose Andresen for assistance.


   Abbreviations
 
AA, amino acid
7AAD, 7-amino-actinomycin-D
BBDP, diabetes-prone BioBreeding rat
BBDR, diabetes resistant BioBreeding rat
C58, C58(NT)D.1.G.OVAR.1
ER, endoplasmic reticulum
Gimap, GTPase of the immunity-associated protein
RNAi, RNA interference
TMRM, tetramethylrhodamine methyl ester
wt, wild type

   Notes
 
Transmitting editor: A. Cooke

Received 6 October 2006, accepted 18 January 2007.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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